Sphingolipids have long been known to be important structural components of the membranes of all mammalian cells. Only recently has it been widely appreciated that sphingolipids are also reservoirs of the bioactive metabolites, ceramide, sphingosine, and sphingosine-1-phosphate, which are now known to act as signaling molecules that regulate many vital biological processes, including cell growth, death, and differentiation. In a continuing and highly successful collaboration with Spiegel's lab at Virginia Commonwealth University, we have begun to elucidate the mechanisms by which these sphingolipid metabolites are produced, how their levels are regulated, and how they mediate their diverse actions. These findings will be important in the future as these sphingolipid metabolites have been shown to be involved in a host of normal and abnormal physiological human processes and diseases, including cancer, asthma, immunological disorders and infectious diseases, and neurodegeneration. For example, we found that intracellularly generated sphingosine-1-phosphate by sphingosine kinase type 1, an enzyme originally cloned by us, may play an important role in breast cancer progression by regulating tumor cell growth and survival. In addition, in work which is related to asthmatic responses and recently submitted for publication, we found that activation of mast cells not only increased cellular levels of sphingosine-1-phosphate, it also induced degranulation of RBL 2H3 and bone marrow derived mast cells likely acting by transactivation of specific mast cell sphingosine-1-phosphate receptors. During the last fiscal year, we also reported two important discoveries that have broad reaching implications for the functions of sphingosine-1-phosphate and the kinases responsible for its formation. We recently cloned a second sphingosine kinase isozyme, type 2, which is larger than type 1 and has different patterns of tissue and developmental expression, suggesting that it might have different functions. Indeed, we have now found that while sphingosine kinase type 1 promotes cell growth and survival, sphingosine type 2 inhibits cell growth and induces cell death. How these two very closely related and similar enzymes that use the same substrate and produce the same product can have such opposite effects and their importance in regulating cell functions is the subject of intense current investigation in our lab. We and others have previously established that the most important actions of sphingosine-1-phosphate are mediated by binding to a family of five specific G protein coupled receptors. These receptors are ubiquitously though not uniformly expressed and are coupled to a wide variety of G proteins and associated signaling pathways, accounting for the ability of sphingosine-1-phosphate to regulate such diverse processes. However, while we have previously suggested that sphingosine-1-phosphate might also have direct intracellular actions, this concept has not been widely accepted due to the scarcity of definitive evidence. We have now found that in contrast to treatment of cells with sphingosine-1-phosphate which increases proliferation by binding to sphingosine-1-phosphate receptors on the cell surface, overexpression of sphingosine kinase type 1 and increased intracellular sphingosine-1-phosphate levels stimulated cell growth and promoted survival independently of these receptors. Hence, exogenous and intracellularly generated S1P can affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent, intracellular function of S1P, reminiscent of its action in yeast that lack sphingosine-1-phosphate receptors. Our contributions to the field were recognized by invitations to write reviews of our work from several prestigious journals, including Nature Review Molecular Cell Biology. We previously found that the sphingolipid metabolites ceramide and sphingosine-1-phosphate also play important roles in regulating tetrahydrobiopterin synthesis as well as survival of neuronal and other types of cells. In collaborative studies with Dr. U. Kang, University of Chicago aimed at developing a new treatment for Parkinson?s disease, we found that mutations could be introduced in tyrosine hydroxylase that prevented feedback inhibition. Cells genetically modified to express this mutant tyrosine hydroxylase together with tetrahydrobiopterin, the required cofactor for formation of DOPA and dopamine from tyrosine, produced much greater levels of dopamine. Our results illustrate the importance of careful consideration of biochemical pathways and interactions between multiple genes in gene therapy. We also found in collaboration with Dr. Z.S. Katusic from the Mayo Clinic that treatment with vitamin C could restore defective endothelial NOS activity in apoE-deficient mice. Moreover, this significantly improved their vascular endothelial function. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of wild type mice. Thus, the beneficial effects of vitamin C on vascular endothelial function appears to be mediated in part by increasing tetrahydrobiopterin and restoration of eNOS enzymatic activity.